The present invention relates to non-anaphylactic, i.e. hypoallergenic, forms of the major timothy grass pollen allergen Phl p 6 and the use of the forms for hyposensitization and for diagnosis. The invention also relates to a method for hyposensitization of a mammalian individual, typically a human individual, suffering from type I allergy against the Phl p 6 allergen.
This application contains a Substitute Sequence Listing located after the abstract. The paper copy of the Substitute Sequence Listing is identical to the computer readable form of the Substitute Sequence Listing except that it lacks formatting.
Type I allergy is a genetically determined hypersensitivity disease that affects more than 20% of the population in industrialized countries [Kay, A. B., Allergy and Allergic Diseases, 1997]. As a consequence of this immuno-disorder, allergic patients produce IgE antibodies against per se innocuous, mostly air-born proteins from pollen, mites, moulds and animal hair/dander. The symptoms of Type I allergy (allergic rhinitis, conjunctivitis, allergic asthma and anaphylactic shock) result from allergen-induced crosslinking of effector cell (mast cell, basophil)-bound IgE antibodies and subsequent release of inflammatory mediators [Segal, et al., PNAS USA 41:457, 1977]. Since approximately 40% of allergic individuals suffer symptoms following contact with grass pollen, research has concentrated on the characterization of relevant grass pollen allergens by protein and immunochemical methods [Freidhoff et al., Allergy Clin. Immunol. 78:1190, 1986]. While groups of major allergens have been identified as cross-reactive moieties that occur in most grass species [Niederberger et al., J. Allergy Clin. Immunol. 102:258, 1998], nothing was known concerning their nature and biological functions.
The recent application of molecular biological techniques to allergen characterization has revealed the primary structures of allergens and facilitated the production of recombinant allergens for diagnostic and therapeutic purposes [Valenta et al., Curr. Opin. Immunol. 7:751, 1995]. Components of the plant cytoskeleton (e. g., profilin) [Valenta et al., Science 253:557, 1991] as well as calcium-binding pollen proteins [Seiberler et al., EMBO J. 13:3481, 1994] have been identified as relevant allergens. The fact that allergic patients exhibit immediate type reactions upon contact with various unrelated allergen sources thus can be explained by cross-reactivity of their IgE antibodies with ubiquitous allergens. Evidence that group 1 grass pollen allergens belong to a family of cell wall-loosening proteins (expansins) [Shcherban et al., PNAS USA 92:9245, 1995] and grass group 5 allergens may possess RNAse activity [Bufe et al., FEBS Lett. 363:6] has restimulated ideas that the biological function of a given protein may be related to its allergenicity. The recent findings that major grass pollen allergens can either become attached to small sized particles (e. g., group 1 allergens to diesel exhaust [Knox et al., Clin. Exp. Allergy 27:246, 1997]) or may become airbom as small pollen subcompartments (e. g., group 5 allergens in amyloplasts [Suphioglu et al., Lancet 339:569, 1992]) would provide a possible mechanism of how certain allergens may be able to reach the deep airways of patients and to elicit allergic asthma.
Therapy of Type I allergic diseases is currently performed by pharmacological treatment and by specific immunotherapy. Specific immunotherapy has been established already early in this century (Noon, Lancet 1: 1572-1573 (1911)) and involves the systemic application of increasing doses of allergens for extended periods. Although specific immunotherapy is recognized as effective treatment, the occurrence of anaphylactic side effects represents one of the major disadvantages of this therapy. To reduce anaphylactic reactions the use of T-cell epitopes has recently been proposed for allergen specific immunotherapy (Briner et al., PNAS USA 90:7608-7612 (1993), and Norman, Curr. Opin. Immunol. 5 :986-973 (1993)). Allergens harbour a great variety of different T-cell epitopes (Ebner et al., J. Immunol 150:1047-1054 (1993); Joost-van-Neerven et al., J. Immunol. 151:2326-2335 (1993); and Schenket al., J. Allergy Clin. Immunol. 96:986-996 (1995)) which may overlap with continuous IgE-epitopes. To prevent crosslinking of effector cell (mast cell, basophil) bound IgE and mediator release, T-cell epitopes and IgE epitopes need to be dissected.
Vrtala et al., J Clin. Invest. 99 [Seiberler et al., EMBO J. 13:3481, 1994]1673-1681 (1997) and WO 99/16467 disclose a novel strategy of reducing the anaphylactic activity of the major birch allergen Bet v 1 by disrupting the three dimensional structure by expressing two parts of the Bet v 1 cDNA representing amino acids 1-74 and 75-160 in Escherichia coli. In contrast to the complete recombinant Bet v 1, the recombinant fragments showed almost no allergenicity. Both non-anaphylactic fragments induced proliferation of human Bet v 1-specific T cell clones, indicating that they harboured all dominant T cell epitopes and therefore could be used for safe and specific T cell immunotherapy. The success of this strategy was believed to be due to the fact that the Bet v 1 allergen possesses discontinuous (i.e. conformational) IgE epitopes rather than continuous IgE epitopes as is the case for many other allergens.
In contrast to the major birch allergen Bet v 1, the major timothy grass pollen allergen Phl p 6 contains continuous (sequential) IgE epitopes and would therefore not be susceptible to the above fragmentation strategy to reduce anaphylactic activity as outlined above.
The present invention provides a hypoallergenic immunogenic molecule derived from the Phl p 6 allergen, wherein the Phl p 6 molecule has an N-terminal and/or C-terminal deletion which makes the molecule at least lack IgE binding capacity.
The present invention also provides a hypoallergenic immunogenic combination of molecules derived from the Phl p 6 allergen, comprising (i) a Phl g 6 molecule having an N-terminal deletion which makes the molecule at least lack IgE binding capacity, and (ii) a Phl p 6 molecule having a C-terminal deletion which makes the molecule at least lack IgE binding capacity, which two molecules together contains the complete sequence of Phl p 6.
The present invention further provides a method for the hyposensitization of a mammal suffering from IgE mediated allergy against a protein allergen.